The plasma kininogens are single-chain glycoproteins which are present in human blood plasma and tissues in two forms: high molecular weight kininogen (120 kDa) and low molecular weight kininogen (64 kDa). The gene that controls the synthesis of both kininogens is the same (Kitamura, N. et al, Nature 305, 545, 1983). The difference between the high molecular weight form and the low molecular weight form is the addition of an unique light chain by a posttranscriptional modification on each of the molecules. The presence of a 56 kDa light chain on high molecular weight kininogen gives this form of kininogen unique antigenic as well as functional properties. The plasma concentration of high molecular weight kininogen is 0.67 micromolar, while the concentration of low molecular weight kininogen is 2.4 micromolar.
The functions of the plasma kininogens are as follows. Both high molecular weight and low molecular weight kininogen are parent molecules for the decapeptide, bradykinin, the most potent naturally-occurring vasodilitory substance. Bradykinin is best liberated from high molecular weight kininogen by plasma kallikrein, but low molecular weight kininogen is a better substrate for tissue kallikreins to release bradykinin.
High molecular weight kininogen also functions as a cofactor for the activation of the following plasma zymogens: factor XII, prekallikrein, and factor XI. These three plasma zymogens/enzymes, along with high molecular weight kininogen, comprise the proteins of the contact phase of plasma proteolysis. In addition to being a cofactor for activation of each of these plasma zymogens, high molecular weight kininogen is also a substrate of each of their proteolytic forms. Moreover, high molecular weight kininogen serves to order the activation and inhibition of these proteases since 75% of plasma prekallikrein and 90% of plasma factor XI circulate in plasma in complex with high molecular weight kininogen. The proteins of the contact phase of plasma proteolysis are a group of proteins that link the plasma proteolytic systems of coagulation, fibrinolysis, complement activation, and blood pressure control.
Low molecular weight kininogen, the preferred substrate for the tissue kallikreins, has recently been identified as being identical to alpha cysteine protease inhibitor--a major plasma inhibitor of cystine proteases (Ohkubo, I. et al, Biochem. 23, 3891, 1984). Since the heavy chain of both high molecular weight and low molecular weight kininogen are identical, high molecular weight kininogen has also been shown to inhibit cysteine proteases (Muller-Esterl, W., FEBS 182, 310, 1985; Sueyoshi, T. et al, FEBS 182, 193, 1985). High molecular weight kininogen has been shown in our laboratories to be the most potent inhibitor of the calcium-activated cysteine protease from platelets (Schmaier, A. H. et al, Blood 66 (Suppl):313A, 1985).
Using high molecular weight kininogen as an example, the molecular structure of the kininogen molecules, as the structure relates to the function of the molecule, can be described. When the intact 120 kDa high molecular weight kininogen molecule is cleaved by plasma kallikrein, the initial two cleavages results in the liberation of the 1000 molecular weight peptide bradykinin, leaving a residual twochain molecule joined together by disulfide bonds. A residual heavy chain (64 kDa) is identical to the heavy chain of plasma low molecular weight kininogen (alpha cysteine protease inhibitor) which can function as a cysteine protease inhibitor. Likewise, an intermediate 56 kDa light chain is formed which contains the unique antigenic and functional properties of the high molecular weight kininogen molecule. This light chain serves as the site for the surface-mediated activities of the molecule that are involved in activating and regulating inhibition of the contact phase zymogens: factor XII, prekallikrein and factor XI. Further cleavage of the 56 kDa light chain of high molecular weight kininogen by kallikrein results in a stable 46 kDa light chain with similar functional properties.
It is hypothesized, but not yet conclusively shown, that the role of plasma kininogens as cysteine protease inhibitors limits the extent of tissue injury upon cytolytic destruction of cells by cysteine proteases. This type of injury occurs as result of complement activation and mechanical destruction. Purified plasma kininogen could be used as a therapeutic tool in the treatment of such conditions as peridontal disease and intervertebral disc disease, where cysteine proteases are pathogenic in these disorders.
The surface-mediated activation of the proteins of the contact phase of plasma proteolysis (factors XI and XII; prekallikrein; high molecular weight kininogen) occurs in pathologic states. Gram negative sepsis, typhoid fever, and acute attacks of hereditary angioedema are serious disease states wherein these proteins are altered and the contact activation system is activated. Early detection of activation of the contact system proteins could have prognostic and therapeutic results on the outcome of treating the above conditions. The ability to develop means for early detection of activation of the contact system proteins can be developed by use of antigenic assays to activation fragments of these proteins.
Kohler and Milstein (Nature 256, 493-497, 1975) were the first to describe the fusion of myeloma cells to immune spleen cells from mice to generate continuous cell lines. These hybrid cell lines, or hybridomas, have characteristics that neither the parental myeloma cells nor parental immune spleen cell possess. Hybridomas are capable of continuously producing homogeneous (monoclonal) antibodies. Prior to the work of Kohler and Milstein, only polyclonal antisera could be obtained.
Although techniques for the production of hybridomas are now extensively described in the literature, e.g., Monoclonal Antibodies, Hybridomas: A New Dimension In Biological Analysis, R. H. Kennet, T. J. McKearn, and K. B. Bechtol, eds., Plenum Press, N. Y. and London (1980), there is no general method for obtaining successful monoclonal antibody-producing hybridomas which can be used with all antigens. Fusion techniques must be varied in each case to obtain hybridomas producing monoclonal antibody to the desired antigen. In order to obtain antibodies specific to a single antigen, laborious purification techniques are required to provide highly purified antigen for immunization. The production of monoclonal antibodies for any given antigen is still a highly empirical process.
The kininogens have been detected in blood by means of coagulant and immunochemical assays using polyclonal antisera. There have been no reports of monoclonal antibodies against kininogens prior to the present invention. The dearth of literature accounts of monoclonal antibodies to the kininogens is no doubt due to difficulties in the purification of antigen and/or the lack of success in preparation of suitable hybridomas.
Hereinafter, "human kininogen", shall mean, unless otherwise indicated, both the high and low molecular weight forms of any kininogen molecule, in all its various forms derived from human plasma, platelets, kidney, skin, leukocytes or other tissues or organs, regardless of whether found in the fluid or the tissue phase.
"HMWK" shall mean high molecular weight human kininogen.
"LMWK" shall mean low molecular weight human kininogen, also known as alpha cysteine protease inhibitor, or alpha.sub.1 -thiol protease inhibitor, or alpha.sub.2 -thiol protease inhibitor.
"Heavy chain" shall mean, when referring or relating to human kininogen, the 64 kDa kallikrein-cleavage fragment of HMWK, said fragment being identical to the heavy chain of LMWK.
"Light chain" shall mean, when referring or relating to human kininogen, the 56 kDa intermediate kallikrein-cleavage fragment of HMWK which has the ability to correct the coagulant defect in total kininogen-deficient plasma.